It has become increasingly clear that the workings of the thymus include greater complexity than first appreciated. Not only is this organ a generator of CD4 and CD8 T cells, but it is also a site for development of highly specialized lineages of natural killer T (NKT) cells, intraepithelial T lymphocytes, regulatory T (Treg) cells, and innate-like CD8 T cells. The detailed molecular mechanisms that control the differentiation of these distinct T cell sublineages from common precursors remain to be determined. Here we focus on the role of TOX (thymocyte selection-associated HMG box protein) in this context, a nuclear DNA-binding protein first identified in our laboratory as a regulator of thymic selection events. We produced TOX-deficient mice, demonstrating that TOX is required for development of the canonical CD4 T cell lineage as well as NKT and Treg cell development. Inhibition of the specific stage of T cell development that is affected in these mutant animals has not been reported previously, allowing novel insights into the process. In addition, we have found that TOX plays roles outside the thymus, including an obligatory role in NK cell development and lymph node organogenesis. We propose here three specific aims to delineate the role and mechanism of action of TOX in development of the immune system. First, we will investigate the relationship between TOX and regulation of CD4 and ThPOK expression in the thymus, the latter a dominant CD4 lineage commitment factor. In addition, we propose a novel approach to identify TOX-dependent genes involved in establishment of the CD4 lineage gene program, and will compare the role of TOX in development of different T cell lineages. Second, we will analyze the role of TOX in development of lymph node organogenesis, focusing on the potential for TOX to regulate lymphoid tissue inducer cell development. We also have replicated the inhibition of NK cell development in the absence of TOX in a culture system. We propose detailed analysis of the role for TOX in this context, including whether TOX might function to regulate another nuclear factor that shares some functional properties with TOX. Third, we propose state-of-the-art approaches to identify direct gene targets and protein binding partners of TOX, in an in vivo context of CD4 T cell development. TOX is highly conserved between rodents and humans, as is its expression pattern. Thus, these studies should provide insight into workings of the human immune system as well. TOX defines a small protein subfamily that includes three other members, one of which has been implicated as a breast cancer susceptibility locus, while TOX itself is highly expressed in some cancer cell lines and tumors. Understanding the mechanism of action of TOX is therefore likely to impact both human health and disease.